负载U266细胞抗原的树突状细胞诱导T细胞的抗骨髓瘤活性

本研究探讨负载骨髓瘤U266细胞裂解物的树突状细胞（DC）瘤苗活化的T细胞对U266细胞的体外杀伤作用。用密度梯度离心法分离外周血单个核细胞,然后分别用含重组人粒细胞-巨噬细胞集落刺激因子（GM-CSF）、重组人白细胞介素-4（IL-4）的AIM-V无血清培养液和含胎牛血清的RPMI1640培养液体外诱导培养DC,用MTT法检测负载U266细胞全抗原的DC活化的T细胞对U266细胞的杀伤作用。结果表明：健康人外周血来源的单个核细胞用无血清培养液和含血清培养液诱导培养均可得到足够数量的DC,这两种培养液培养的DC在表型上无明显差异。分别用负载U266细胞全抗原的DC＋IL-2、成熟DC＋IL-2和单独应用IL-2刺激后的T细胞与U266细胞共培养,其对U266细胞的杀伤作用逐渐减弱,负载U266细胞全抗原的DC＋IL-2刺激后的T细胞较成熟DC＋IL-2刺激后的T细胞对U266细胞的杀伤作用明显,且T细胞对U266细胞的杀伤率随效靶比的增加而升高。结论：用GM-CSF、IL-4诱导培养健康人外周血单个核细胞可以得到不成熟的DC,体外负载U266细胞全抗原的DC可以刺激自体T细胞增殖并能杀伤U266细胞。     

抗原致敏、GM-CSF基因修饰的树突状细胞诱导免疫杀伤多发性骨髓瘤细胞U266的研究

本研究探讨负载有多发性骨髓瘤细胞U266可溶性抗原并携带外源基因粒-巨噬细胞集落刺激因子（GM—CSF）的树突状细胞（DC）,在体外激活自身T淋巴细胞形成细胞毒T淋巴细胞（CTLs）对U266细胞的杀伤作用。用U266可溶性抗原致敏DC,再用含有外源基因GM—CSF的腺病毒感染DC,将所得的DC与T细胞混合培养以形成对U266具有特异杀伤作用的CTL,最后通过测定乳酸脱氢酶（LDH）以计算CTL对U266的杀伤率。结果表明：负载抗原及外源基因组、负载抗原组和对照组之间的杀伤率存在显著差异（n=3,F=10．939,P〈0．05）;两两比较表明,负载抗原及外源基因组的杀伤率高于其它两组（P〈0．001）,且负载抗原组高于对照组（P〈0．001）。结论：以U266可溶性抗原致敏的DC可诱导出对U266具有特异杀伤作用的CTL,当所致敏的DC通过腺病毒感染而带有外源基因GM—CSF时,所诱导的细胞毒杀伤反应则进一步增强。     

负载舌鳞癌抗原的树突状细胞抗肿瘤的实验研究

目的探讨针对舌鳞癌的特异性免疫治疗作用。方法培养舌鳞癌细（Tca8113），43℃热后提取抗原并与树突状细胞（DC）共同培养，检测其对舌鳞癌细胞的特异性杀伤活性。结果负载抗原的DC对舌鳞癌细胞的杀伤活性明显高于未负载抗原的DC。结论DC负载抗原后可明显提高其抗肿瘤活性。     

抗原负载的DC及CIK对结肠癌细胞SW1116杀伤活性的比较研究

目的 探讨树突状细胞（dendritic cells,DCs）以及CIK（cytokine induced cells）细胞对结肠癌细胞SW1116的体外杀伤作用,为结肠癌的治疗提供新的治疗手段.方法 用IL-4、GM-CSF、TNFα等细胞因子诱导培养DC细胞,并用抗原进行负载;用CD3Ab、IFNγ、IL -2、IL-1α等诱导培养CIK细胞,分别用抗原负载的DC、CIK细胞对人结肠癌细胞SW1116进行体外杀伤活性实验,比较两者对SW1116的杀伤效果.结果 抗原负载的DC和CIK对SW1116均有较强的杀伤效果,分别达到64.10%和67.70%.结论 抗原负载的DC及CIK对人结肠癌细胞SW1116在体外具有较强的杀伤作用.     

负载肿瘤抗原的DC疫苗体外诱导的特异性抗膀胱癌效应研究

目的探讨负载膀胱癌抗原成分树突状细胞（dendritic cells,DC）疫苗的制备和体外诱导T淋巴细胞特异性杀伤膀胱癌细胞的作用。方法冻融法制备EJ细胞裂解物抗原成分,体外培养的人外周血单个核细胞（hu-PBMC）在rhGM-CSF、rhIL-4、TNF-α诱导下分化出DC,负载EJ细胞裂解物抗原后制备膀胱癌DC疫苗;免疫磁珠分离法从人免疫重建Balb/c裸小鼠脾脏组织中分离CD3＋T淋巴细胞,3H-TdR掺入试验测定DC疫苗刺激自体T淋巴细胞增殖的能力,51Cr释放试验检测DC疫苗诱导的T细胞对EJ细胞的杀伤作用。结果 Hu-PBMC在细胞因子rhGM-CSF、rhIL-4和TNF-α的刺激下分化为成熟DC,负载EJ抗原的DC疫苗体外可使同源T淋巴细胞活化,增殖指数增加,活化的T淋巴细胞对EJ细胞的杀伤率为（62.58±6.13）%,和对照组比较差异有统计学意义（P〈0.05）。结论负载膀胱癌冻融抗原的DC疫苗体外可诱导人T淋巴细胞活化增殖,对EJ细胞有明显的杀伤作用。     

卵巢癌抗原冲击DC在体外对SKOV3细胞作用的研究

目的探讨卵巢癌患者外周血和腹水中培养的树突细胞(DC)在体外激活淋巴细胞产生的肿瘤特异性细胞毒T淋巴细胞(CTL)作用,及其对卵巢癌SKOV3细胞的杀伤作用。方法分别培养卵巢癌患者外周血及腹水来源DC,进行细胞形态学观察及细胞表面分子表达检测;以卵巢癌冻融抗原致敏DC,检测两种来源DC刺激T淋巴细胞增殖的能力及对IL-2水平的影响;检测抗原负载DC与T细胞诱导CTL对卵巢癌细胞的杀伤作用。结果镜下显示卵巢癌患者外周血及腹水来源的DC形态学无明显差异,腹水来源DC成熟时间较外周血来源DC晚,二者表面分子表达的阳性率除CD83外无明显差异;卵巢癌冻融抗原负载DC上调其表面相关分化抗原表达,统计学差异具有显著性(P0.05);经卵巢癌冻融抗原负载后的DC刺激T淋巴细胞的增殖,差异有显著性(P0.05),但两种来源DC间刺激指数无统计学差异;抗原负载DC与无抗原负载DC相比,其上清中IL-12浓度增高,差异具有显著性(P0.05);负载与不负载抗原的DC对卵巢癌细胞杀伤作用增强,与对照组相比具有显著性差异(P0.05),随着效应细胞数量的增高,杀伤作用增强,加入IL-2增强其杀伤作用,但外周血及腹水来源DC间相应情况下对卵巢癌细胞的杀伤作用无统计学差异(P0.05)。结论卵巢癌患者外周血及腹水来源的DC均具有诱导CTL作用,DC作为载体负载肿瘤抗原后可以激发T淋巴细胞形成CTL,在体外有效的杀伤卵巢癌SKOV3细胞。     

抗原致敏外周血树突状细胞治疗脑胶质瘤的实验研究

目的 观察树突状细胞(DCs)从凋亡脑胶质瘤细胞获取抗原后,体外诱导抗肿瘤免疫应答及对胶质瘤细胞的特异性免疫杀伤效果.方法 用粒-巨噬细胞集落刺激因子(GM-CSF)加白介素-4(IL-4)从人外周血分化、诱导DCs、γ-射线在体外诱导培养的人脑胶质瘤细胞凋亡,将DCs、T淋巴细胞和凋亡胶质瘤细胞共培养,同时设计不同类型肿瘤细胞(U937及培养胶质瘤细胞)作对照,分离、富集DCs、T淋巴细胞进行免疫应答及肿瘤细胞杀伤试验.结果 与凋亡胶质瘤细胞共培养之Dcs可以有效提呈胶质瘤细胞抗原,有强烈的免疫应答,刺激的细胞毒T淋巴细胞((CTLs)特异性杀伤胶质瘤细胞.结论 用GM-CSF加IL-4从人外周血分化、诱导的DCs能从凋亡胶质瘤细胞有效提呈肿瘤抗原并诱导出显著的杀伤胶质瘤细胞的免疫反应,可望成为有效的肿瘤特异性免疫治疗新途径.     

青蒿琥酯诱导凋亡的U937细胞负载树突状细胞后的免疫应答

本研究探讨凋亡白血病细胞负载的树突状细胞（dendritic cells,DC）能否诱导特异性细胞毒T淋巴细胞反应。用青蒿琥酯诱导U937细胞凋亡,从正常人外周血单个核细胞诱生DC,将凋亡U937细胞负载DC,并添加TNF-α诱导DC成熟,然后与自体T淋巴细胞共育,并联合IL-2以诱生细胞毒性T淋巴细胞（CTL）;应用流式细胞术分析DC和T细胞免疫表型,采用Dextran-FITC内吞试验检测DC的抗原摄取能力;用ELISA法检测IL-12p70的产生;采用MTT法检测凋亡U937细胞负载及未负载DC诱导的自体T淋巴细胞对不同的靶细胞（U937细胞和NB4细胞）的杀伤效应。结果表明：青蒿琥酯1μg/ml作用于U937细胞48小时后,U937细胞的凋亡率达51.52%,DC在未成熟阶段时吞噬Dextran-FITC的能力最强,负载凋亡U937细胞之后并不能促使未成熟DC（iDC）分化为成熟DC（mDC）。凋亡U937细胞负载后的iDC在TNF-α的作用下能够成为mDC。与未负载的mDC相比,凋亡U937细胞负载后的mDC（mDC-^ApoU937）分泌IL-12p70水平明显增高,而且由其所激发和扩增的T细胞以CD8^＋的T细胞为主。细胞毒性实验显示：mDC-^ApoU937诱导的T细胞的对U937细胞的杀伤显著超过对NB4细胞的杀伤。结论：mDC-^ApoU937能有效地诱导特异性抗白血病效应。     

顺铂联合血管内皮抑制素对体外培养的U251人胶质瘤细胞生长的影响

目的以体外培养的U251人胶质瘤细胞作为模型,观察顺铂联合血管内皮抑制素对肿瘤细胞生长的影响。方法采用不同浓度的顺铂和血管内皮抑制素及顺铂与血管内皮抑制素的组合作用于体外培养的U251人胶质瘤细胞,以MTT比色法检测其对肿瘤细胞生长的影响。结果不同浓度的顺铂与血管内皮抑制素联合应用对体外培养的U251人胶质瘤细胞生长具有非常明显的协同抑制作用,强于单独使用顺铂或血管内皮抑制素组。结论顺铂联合血管内皮抑制素对体外培养的U251人胶质瘤细胞具有协同杀伤作用。     

热休克蛋白70激发树突状细胞抗肝癌作用的实验研究

肿瘤的治疗除了手术、放疗、化疗外,另一个不可忽视的疗法就是免疫治疗,近年来它在肿瘤治疗中的作用越来越受到重视。本实验通过在体外用脐带血诱导树突状细胞（DC）,并用由肝癌细胞SMMC-7721提取的热休克蛋白70-肿瘤肽复合物（HSP70-PC）负载DC,检测其对肝癌细胞的杀伤作用,证明此活化DC对肿瘤细胞的特异性杀伤活性,为临床应用提供实验依据。     

自杀基因疗法结合HSP-PC瘤苗抗黑色素瘤研究

目的 :研究 5 -氟尿嘧啶 /胞嘧啶脱氨酶 (5FC/CD)基因疗法与热休克蛋白 -多肽复合物 (HSP -PC)瘤苗免疫疗法联合抗肿瘤效果。方法 :将携带CD基因的重组腺病毒注射到小鼠黑色素瘤体内 ,腹腔注射 5 -FC ,同时皮下接种HSP70 -PC。结果 :经联合治疗后 ,70 %荷瘤小鼠肿瘤体积缩小、消退 ,小鼠存活期延长 ,肿瘤组织明显坏死 ,炎症细胞、CD4+ 、及CD8+ T细胞浸润明显。结论 :5 -FC/CD基因疗法结合HSP -PC瘤苗免疫疗法抗小鼠黑色素瘤作用显著 ,具有临床应用前景。     

Comparative analysis of antigen loading strategies of dendritic cells for tumor immunotherapy

Dendritic cells (DCs) loaded with antigens can effectively stimulate host immune responses to syngeneic tumors, but there is considerable controversy as to which forms of antigen-loading are most immunogenic. Here, the authors compared immunotherapeutic reactivities of DCs loaded with a variety of antigen preparations. Because DC maturation stages affect their capacities of antigen processing and presentation, two DC populations were used for the current analysis: in vivo Flt-3 ligand-induced mature DCs and in vitro bone marrow-derived DCs, which were less mature. To facilitate a direct comparison, the LacZ gene-transduced B16 melanoma model system was used, where beta-galactosidase served as the surrogate tumor-rejection antigen. DC loading strategies included pulsing with the beta-galactosidase protein, H-2K restricted peptide, tumor cell lysate, and irradiated tumor cells and fusion of DCs with tumor cells. Our results demonstrated that electrofusion of DCs and tumor cells generated a therapeutic vaccine far superior to other methods of DC loading. For the treatment of 3-day established pulmonary tumor nodules, a single intranodal vaccination plus IL-12 resulted in a significant reduction of metastatic nodules, while other DC preparations were only marginally effective. Immunotherapy mediated by the fusion cells was tumor antigen-specific. Consistent with their therapeutic activity, fusion hybrids were the most potent stimulators to induce specific IFN-gamma secretion from immune T cells. Furthermore, fusion cells also stimulated a small amount of IL-10 production from immune T cells. However, this IL-10 secretion was also induced by other DC preparations and did not correlate with in vivo therapeutic reactivity.     

Differential immune responses mediated by adenovirus- and lentivirus-transduced DCs in a HER-2/neu overexpressing tumor model

Recent investigations have demonstrated that adenoviral and lentiviral vectors encoding HER-2 can be utilized in cancer immunotherapy. However, it is not known whether both viral systems elicit a similar immune response. Here, we compare the immune response in mice induced by dendritic cells (DCs) infected with either recombinant adenovirus or lentivirus encoding rat HER-2 (rHER-2). Both vaccine types yielded similar control of tumor growth, but we found clear differences in their immune responses 10 days after DC immunization. Adenovirus rHER-2-transduced DCs elicited locally and systemically high frequencies of CD4+ and CD8+ T cells, while lentivirus rHER-2-transduced DCs predominantly led to CD4+ T-cell infiltration at the tumor site. Splenocytes from mice immunized with lentivirus rHER-2-transduced DCs secreted higher levels of interferon (IFN)-γ, mainly by CD4+ T cells, following stimulation by RM-1-mHER-2 tumors. In contrast, the adenovirus vaccinated group exhibited CD4+ and CD8+ T cells that both contributed to IFN-γ production. Besides an established cellular immune response, the rHER-2/DC vaccine elicited a significant humoral response that was highest in the adenovirus group. DC subsets and regulatory T cells in the spleen were also differentially modulated in the two vaccine systems. Finally, adoptive transfer of splenocytes from both groups of immunized mice strongly inhibited in vivo tumor growth. Our results suggest that not only the target antigen but also the virus system may determine the nature and magnitude of antitumor immunity by DC vaccination.     

Immunomodulatory dendritic cells generated from nonfractionated bulk peripheral blood mononuclear cell cultures induce growth of cytotoxic T cells against renal cell carcinoma

Dendritic cells (DCs) loaded with tumor antigens have the potential to become a powerful tool for clinical cancer treatment. Recently, the authors showed that a tumor-specific immune response can be elicited in culture via stimulation with autologous renal tumor lysate (Tuly)-loaded DCs that were generated from cytokine-cultured adherent peripheral blood mononuclear cells (PBMCs). Here, the authors show that immunomodulatory DCs can be generated directly from nonfractionated bulk PBMC cultures. Kinetic studies of DC differentiation and maturation in PBMC cultures were performed by monitoring the acquisition of DC-associated molecules using fluorescence-activated cell sorting analysis to determine the percentage of positive immunostained cells and the mean relative linear fluorescence intensity (MRLFI). Compared with conventional adherent CD14+ cultures, which have mostly natural killer, T, and B cells removed before cytokine culture, bulk PBMC cultures exhibited an early loss of CD14+ cells (day 0 = 78.8%, day 2 = 29.6% versus day 0 = 74%, day 2 = 75%) with an increase in yield of mature DCs (DC19- CD83+) (day 5 = 17%, day 6 = 21%, day 7 = 22% versus day 5 = 11%, day 6 = 15%, day 7 = 23%). Although a comparable percentage of DCs expressing CD86+ (B7-2), CD40+, and HLA-DR+ were detected in both cultures, higher expression levels were detected in DCs derived from bulk culture (CD86 = MRLFI 3665.1 versus 2662.1 on day 6; CD40 = MRLFI 1786 versus 681.2 on day 6; HLA-DR = MRLFI 6018.2 versus 3444.9 on day 2). Cytokines involved in DC maturation were determined by polymerase chain reaction demonstrating interleukin-6 (IL-6), IL-12, interferon-gamma, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha mRNA expression by bulk culture cells during the entire 9-day culture period. This same cytokine mRNA profile was not found in the conventional adherent DC culture. Autologous renal Tuly (30 micrograms protein/10(7) PBMCs) enhanced human leukocyte antigen expression by DCs (class I = 7367.6 versus 4085.4 MRFLI; class II = 8277.2 versus 6175.7 MRFLI) and upregulated cytokine mRNAs levels. Concurrently, CD3+ CD56-, CD3+ CD25+, and CD3+ TCR+ cell populations increased and cytotoxicity against autologous renal cell carcinoma tumor target was induced. Specific cytotoxicity was augmented when cultures were boosted continuously with IL-2 (20 U/mL biological response modifier program) plus Tuly stimulation. These results suggest that nonadherent PBMCs may participate in enhancing DC maturation. Besides the simplicity of this culture technique, bulk DC cultures potentially may be used with the same efficiency as conventional purified DCs. Furthermore, bulk culture-derived DCs may be used directly in vivo as a tumor vaccine, or for further ex vivo expansion of co-cultured cytotoxic T cells to be used for adoptive immunotherapy.     

Potential approaches for more successful dendritic cell-based immunotherapy

Introduction: Dendritic cells (DCs) are the most important antigen-presenting cell population for activating antitumor T-cell responses; therefore, they offer a unique opportunity for specific targeting of tumors.

Areas covered: We will discuss the critical factors for the enhancement of DC vaccine efficacy: different DC subsets, types of in vitro DC manufacturing protocol, types of tumor antigen to be loaded and finally different adjuvants for activating them. We will cover potential combinatorial strategies with immunomodulatory therapies: depleting T-regulatory (Treg) cells, blocking VEGF and blocking inhibitory signals. Furthermore, recommendations to incorporate these criteria into DC-based tumor immunotherapy will be suggested.

Expert opinion: Monocyte-derived DCs are the most widely used DC subset in the clinic, whereas Langerhans cells and plasmacytoid DCs are two emerging DC subsets that are highly effective in eliciting cytotoxic T lymphocyte responses. Depending on the type of tumor antigens selected for loading DCs, it is important to optimize a protocol that will generate highly potent DCs. The future aim of DC-based immunotherapy is to combine it with one or more immunomodulatory therapies, for example, Treg cell depletion, VEGF blockage and T-cell checkpoint blockage, to elicit the most optimal antitumor immunity to induce long-term remission or even cure cancer patients.     

Vaccination with immature dendritic cells combined with CD40 mAb induces protective immunity against B lymphoma in hu-SCID mice

Dendritic cells (DCs) pulsed by tumor antigens have been widely used as tumor vaccines to specifically trigger the cytotoxicity of CD8+ T cells. But the tumor microenviroment with enriched immunosuppressants hampered DC maturation and co-stimulation. CD40/CD40L signaling, one of the most important co-stimulatory molecules is capable of effectively skewing the immune response by promoting DCs maturation and co-stimulation. To establish a novel specific immunotherapeutic approach for the use of DC vaccine in the treatment of B lymphoma, hu-SCID mice bearing B lymphoma were vaccinated by different combination of tumor antigen pulsed DC or imDC vaccines and immune-enhancing agencies such as agonist CD40 mAb and T cells. The results of immature DCs combined with agonistic CD40 mAb were encouraging with achievement of tumor regression and induction of antigen-specific immune responses. These findings demonstrated the potential utility of imDC-based tumor vaccination combining with agonistic CD40 mAb in the treatment of malignant lymphoma.     

Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming

Antigen-pulsed dendritic cells (DCs) are used as natural adjuvants for vaccination, but the factors that influence the efficacy of this treatment are poorly understood. We investigated the parameters that affect the migration of subcutaneously injected mouse-mature DCs to the draining lymph node. We found that the efficiency of DC migration varied with the number of injected DCs and that CCR7+/+ DCs migrating to the draining lymph node, but not CCR7-/- DCs that failed to do so, efficiently induced a rapid increase in lymph node cellularity, which was observed before the onset of T cell proliferation. We also report that DC migration could be increased up to 10-fold by preinjection of inflammatory cytokines that increased the expression of the CCR7 ligand CCL21 in lymphatic endothelial cells. The magnitude and quality of CD4+ T cell response was proportional to the number of antigen-carrying DCs that reached the lymph node and could be boosted up to 40-fold by preinjection of tumor necrosis factor that conditioned the tissue for increased DC migration. These results indicate that DC number and tissue inflammation are critical parameters for DC-based vaccination.     

In vivo vaccination with tumor cell lysate plus CpG oligodeoxynucleotides eradicates murine glioblastoma

Dendritic cell (DC) vaccines have shown antitumor activity in experimental glioma models and in human glioma patients. The typical approach has been to generate the vaccine ex vivo, by pulsing DCs with tumor lysate or peptides, then administering the DCs back into the patient. This process requires significant expertise and expenses in DC generation. Immature DCs which present antigens to T cells in the absence of appropriate costimulatory signals can lead to induction of immune tolerance. Recent studies have shown that coadministration of toll-like receptor 9 agonists, CpG oligodeoxynucleotides, can promote DC vaccines to break immune tolerance to tumor antigens. We investigated the therapeutic efficacy of in vivo DC activation, by directly administering glioma cell lysate with CpG oligodeoxynucleotides (CpG/lysate), in glioma-bearing mice. Subcutaneous vaccination with CpG/lysate induced a significant increase (P<0.05) in the number of total T cells and activated DCs in lymph nodes draining the vaccination site as compared to mice treated with CpG or tumor lysate alone. Mice vaccinated with CpG/lysate exhibited over 2 times greater median survival than mice in the control groups (P<0.05). Up to 55% of mice vaccinated with CpG/lysate were rendered tumor-free as assessed by survival and bioluminescent imaging. Splenocytes taken from mice vaccinated with CpG/lysate elaborated significantly more IFN-gamma production and displayed greater tumor cell lysis activity compared with the control groups (P<0.05). These results suggest direct vaccination with CpG/lysate provides an alternative and effective approach to induce host antitumor immunity and warrants clinical investigation in the immunotherapy of cancer.     

Optimization of dendritic cell loading with tumor cell lysates for cancer immunotherapy

The immune response to cancer is critically determined by the way in which tumor cells die. As necrotic, stress-associated death can be associated with activation of antitumor immunity, whole tumor cell antigen loading strategies for dendritic cell (DC)-based vaccination have commonly used freeze-thaw "necrotic" lysates as an immunogenic source of tumor-associated antigens. In this study, the effect of such lysates on the ability of DCs to mature in response to well-established maturation stimuli was examined, and methods to enhance lysate-induced DC activation explored. Freeze-thaw lysates were prepared from murine tumor cell lines and their effects on bone marrow-derived DC maturation and function examined. Unmodified freeze-thaw tumor cell lysates inhibited the toll-like receptor-induced maturation and function of bone marrow-derived DCs, preventing up-regulation of CD40, CD86, and major histocompatibility complex class II, and reducing secretion of inflammatory cytokines [interleukin (IL)-12 p70, tumor necrosis factor-alpha, and IL-6]. Although IL-10 secretion was increased by lysate-pulsed DCs, this was not responsible for the observed suppression of IL-12. Although activation of the nuclear factor-kappaB pathway remained intact, the kinase activity of phosphorylated p38 mitogen-activated protein kinase was inhibited in lysate-pulsed DCs. Lysate-induced DC suppression was partially reversed in vitro by induction of tumor cell stress before lysis, and only DCs loaded with stressed lysates afforded protection against tumor challenge in vivo. These data suggest that ex vivo freeze-thaw of tumor cells does not effectively mimic in vivo immunogenic necrosis, and advocates careful characterization and optimization of tumor cell-derived vaccine sources for cancer immunotherapy.